Potassium-evoked Glutamate Release Research Articles

Effect of Cocaine on Potassium-Evoked Release of Glutamate From Fetal Rat Brain Synaptosomes.

Introduction Cocaine use during pregnancy can affect fetal brain development. A fetal brain injury could happen from the direct effect of cocaine on the developing brain or from the reduction of placental perfusion from vasoconstriction, which may lead to hypoxia-ischemia. A potential mechanism for brain injury could be due to a neurotransmitter imbalance within the brain, especially glutamate. In an immature rat brain synaptosome model, we explored the additive effect of cocaine alone on glutamate release and the effect of cocaine combined with simulated hypoxic depolarization using potassium as a surrogate. Method Rat pups' brains were dissected and placed on a chilled petri dish. They then entered the experimental protocol. The suspended synaptosomes were divided equally into four experimental groups (control, high potassium "surrogate to hypoxic stimulation," cocaine, and cocaine + high K). Reversed-phase high-performance liquid chromatography analyzed glutamate with fluorescent detection Results The glutamate level was lowest in the cocaine-only group, with a level of 1.96 × 104, compared to the control and high potassium group. However, combining cocaine with high potassium seemed to generate a synergistic effect, achieving the highest glutamate level of all groups with a value of 5.31 × 104. Post hoc Conover's test for multiple pairwise-comparison between groups was done. In comparing various solutions to control, we did not find a statistically significant difference with the cocaine-only solution with a p-value of 0.074. Also, on comparing various other solutions to each other, there was no statistically significant difference between cocaine vs. cocaine + high potassium a p-value of 0.074. Conclusion Our data support the conclusion that cocaine alone does not induce glutamate release from fetal rat brain synaptosomes. Exposure to high potassium does lead to glutamate release. However, cocaine greatly enhances glutamate release in the presence of high potassium levels. This could explain how cocaine affects brain maturation during pregnancy with a low oxygen tension environment in the placenta.This hypothesis should be tested in vivo.

Brain alterations in GABA, glutamate and glutamine markers after chronic atrazine exposure in the male albino rat.

Atrazine (ATR; 2-chloro-4-ethylamino-6-isopropylamino-s-triazine) is an herbicide widely used to kill annual grasses and broadleaf weeds in crops such as corn, sorghum, and sugarcane. Studies in rodents have shown that chronic ATR exposure is associated with alterations in the nigrostriatal dopaminergic pathway such as hyperactivity, decreased striatal dopamine levels, and diminished numbers of tyrosine hydroxylase positive cells in substantia nigra pars compacta. However, the effects of ATR on neurotransmitters such as GABA and glutamate have been scarcely studied. To evaluate the impact of ATR on motor and anxiety tasks, tissue levels of GABA, glutamate, glutamine, and extracellular and potassium-evoked release of glutamate in the striatum, we daily exposed Sprague-Dawley male rats to 1 or 10mg ATR/kg of body weight for 12-14months. As previously reported, chronic ATR exposure causes hyperactivity in the group exposed to 10mg ATR/kg and increased anxiety in both groups exposed to ATR. GABA, glutamate, and glutamine levels were differentially altered in brain regions related to nigrostriatal and mesolimbic systems, the amygdala, and the prefrontal cortex. The groups exposed to 10mg ATR/kg showed increased extracellular levels and release of glutamate in the striatum. These neurochemical alterations could underlie the behavioral changes observed in rats. These results indicate that chronic exposure to the herbicide ATR disrupts the neurochemistry of several brain structures and could be a risk factor for the development of neurodegenerative diseases.

Acute treatment with doxorubicin affects glutamate neurotransmission in the mouse frontal cortex and hippocampus

Doxorubicin (DOX) is a potent chemotherapeutic agent known to cause acute and long-term cognitive impairments in cancer patients. Cognitive function is presumed to be primarily mediated by neuronal circuitry in the frontal cortex (FC) and hippocampus, where glutamate is the primary excitatory neurotransmitter. Mice treated with DOX (25mg/kg i.p.) were subjected to in vivo recordings under urethane anesthesia at 24h post-DOX injection or 5 consecutive days of cognitive testing (Morris Water Maze; MWM). Using novel glutamate-selective microelectrode arrays, amperometric recordings measured parameters of extracellular glutamate clearance and potassium-evoked release of glutamate within the medial FC and dentate gyrus (DG) of the hippocampus. By 24h post-DOX injection, glutamate uptake was 45% slower in the FC in comparison to saline-treated mice. In the DG, glutamate took 48% longer to clear than saline-treated mice. Glutamate overflow in the FC was similar between treatment groups, however, it was significantly increased in the DG of DOX treated mice. MWM data indicated that a single dose of DOX impaired swim speed without impacting total length traveled. These data indicate that systemic DOX treatment changes glutamate neurotransmission in key nuclei associated with cognitive function within 24h, without a lasting impact on spatial learning and memory. Understanding the functional effects of DOX on glutamate neurotransmission may help us understand and prevent some of the debilitating side effects of chemotherapeutic treatment in cancer survivors.

Peripherally restricted viral challenge elevates extracellular glutamate and enhances synaptic transmission in the hippocampus.

Peripheral infections increase the propensity and severity of seizures in susceptible populations. We have previously shown that intraperitoneal injection of a viral mimic, polyinosinic-polycytidylic acid (PIC), elicits hypersusceptibility of mice to kainic acid (KA)-induced seizures. This study was undertaken to determine whether this seizure hypersusceptibility entails alterations in glutamate signaling. Female C57BL/6 mice were intraperitoneally injected with PIC, and after 24h, glutamate homeostasis in the hippocampus was monitored using the enzyme-based microelectrode arrays. PIC challenge robustly increased the level of resting extracellular glutamate. While pre-synaptic potassium-evoked glutamate release was not affected, glutamate uptake was profoundly impaired and non-vesicular glutamate release was augmented, indicating functional alterations of astrocytes. Electrophysiological examination of hippocampal slices from PIC-challenged mice revealed a several fold increase in the basal synaptic transmission as compared to control slices. PIC challenge also increased the probability of pre-synaptic glutamate release as seen from a reduction of paired-pulse facilitation and synaptic plasticity as seen from an enhancement of long-term potentiation. Altogether, our results implicate a dysregulation of astrocytic glutamate metabolism and an alteration of excitatory synaptic transmission as the underlying mechanism for the development of hippocampal hyperexcitability, and consequently seizure hypersusceptibility following peripheral PIC challenge. Peripheral infections/inflammations enhance seizure susceptibility. Here, we explored the effect of peritoneal inflammation induced by a viral mimic on glutamate homeostasis and glutamatergic neurotransmission in the mouse hippocampus. We found that peritoneal inflammation elevated extracellular glutamate concentration and enhanced the probability of pre-synaptic glutamate release resulting in hyperexcitability of neuronal networks. These mechanisms are likely to underlie the enhanced seizure propensity.

Altered neurotransmission prior to cognitive decline in AβPP/PS1 mice, a model of Alzheimer's disease.

Indirect evidence supports altered glutamate signaling with Alzheimer's disease, however, it is not known if glutamate neurotransmission is impacted prior to cognitive decline. We examined cognition and glutamate neurotransmission in 2-4 month AβPP/PS1, an Alzheimer's disease model, and age-matched control mice. There were no differences in learning and memory as assessed by Morris water maze. However, in vivo electrochemical measures of potassium-evoked glutamate release in the CA1, but not the CA3 or dentate, was significantly elevated in AβPP/PS1 mice. These data support changes in the glutamatergic system that precedes cognitive decline in a mouse model of Alzheimer's disease.

P301L tau expression affects glutamate release and clearance in the hippocampal trisynaptic pathway.

Individuals at risk of developing Alzheimer's disease (AD) often exhibit hippocampal hyperexcitability. A growing body of evidence suggests that perturbations in the glutamatergic tripartite synapse may underlie this hyperexcitability. Here, we used a tau mouse model of AD (rTg(TauP301L)4510) to examine the effects of tau pathology on hippocampal glutamate regulation. We found a 40% increase in hippocampal vesicular glutamate transporter, which packages glutamate into vesicles, and has previously been shown to influence glutamate release, and a 40% decrease in hippocampal glutamate transporter 1, the major glutamate transporter responsible for removing glutamate from the extracellular space. To determine whether these alterations affected glutamate regulation in vivo, we measured tonic glutamate levels, potassium-evoked glutamate release, and glutamate uptake/clearance in the dentate gyrus, cornu ammonis 3(CA3), and cornu ammonis 1(CA1) regions of the hippocampus. P301L tau expression resulted in a 4- and 7-fold increase in potassium-evoked glutamate release in the dentate gyrus and CA3, respectively, and significantly decreased glutamate clearance in all three regions. Both release and clearance correlated with memory performance in the hippocampal-dependent Barnes maze task. Alterations in mice expressing P301L were observed at a time when tau pathology was subtle and before readily detectable neuron loss. These data suggest novel mechanisms by which tau may mediate hyperexcitability. Pre-synaptic vesicular glutamate transporters (vGLUTs) package glutamate into vesicles before exocytosis into the synaptic cleft. Once in the extracellular space, glutamate acts on glutamate receptors. Glutamate is removed from the extracellular space by excitatory amino acid transporters, including GLT-1, predominantly localized to glia. P301L tau expression increases vGLUT expression and glutamate release, while also decreasing GLT-1 expression and glutamate clearance.

Striatal Glutamate Release in l-DOPA-Induced Dyskinetic Animals

l-DOPA-induced dyskinesia is a common side effect developed after chronic treatment with 3,4-dihydroxyphenyl-l-alanine (l-DOPA) in Parkinson's disease. The biological mechanisms behind this side effect are not fully comprehended although involvement of dopaminergic, serotonergic, and glutamatergic systems has been suggested. The present study utilizes in vivo amperometry to investigate the impact from unilateral 6-hydroxydopamine lesions and l-DOPA (4 mg/kg, including benserazide 15 mg/kg) -induced dyskinetic behavior on striatal basal extracellular glutamate concentration and potassium-evoked glutamate release in urethane-anesthetized rats. Recordings were performed before and after local l-DOPA application in the striatum. In addition, effects from the 5-HT1A receptor agonist (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OHDPAT; 1 mg/kg) was assessed on glutamate release and on dyskinetic behavior. The results revealed a bilateral ∼30% reduction of basal extracellular glutamate concentration and attenuated potassium-evoked glutamate release after a unilateral dopamine-depletion in l-DOPA naïve animals. In dyskinetic subjects, basal glutamate concentration was comparable to normal controls, although potassium-evoked glutamate release was reduced to similar levels as in drug naïve dopamine-lesioned animals. Furthermore, acute striatal l-DOPA administration attenuated glutamate release in all groups, except in the dopamine-lesioned striatum of dyskinetic animals. Co-administration of 8-OHDPAT and l-DOPA decreased dyskinesia in dopamine-lesioned animals, but did not affect potassium-evoked glutamate release, which was seen in normal animals. These findings indicate altered glutamate transmission upon dopamine-depletion and dyskinesia.

Reduction of vesicle-associated membrane protein 2 expression leads to a kindling-resistant phenotype in a murine model of epilepsy

Our previous work has correlated permanent alterations in the rat neurosecretory machinery with epileptogenesis. Such findings highlighted the need for a greater understanding of the molecular mechanisms underlying epilepsy so that novel therapeutic regimens can be designed. To this end, we examined kindling in transgenic mice with a defined reduction of a key element of the neurosecretory machinery: the v-SNARE (vesicle-bound SNAP [soluble NSF attachment protein] receptor), synaptobrevin/vesicle-associated membrane protein 2 (VAMP2). Initial analysis of biochemical markers, which previously displayed kindling-dependent alterations in rat hippocampal synaptosomes, showed similar trends in both wild-type and VAMP2+/− mice, demonstrating that kindled rat and mouse models are comparable. This report focuses on the effects that a ∼50% reduction of synaptosomal VAMP2 has on the progression of electrical kindling and on glutamate release in hippocampal subregions. Our studies show that epileptogenesis is dramatically attenuated in VAMP2+/− mice, requiring both higher current and more stimulations to reach a fully kindled state (two successive Racine stage 5 seizures). Progression through the five identifiable Racine stages was slower and more variable in the VAMP2+/− animals compared with the almost linear progression seen in wild-type littermates. Consistent with the expected effects of reducing a major neuronal v-SNARE, glutamate-selective, microelectrode array (MEA) measurements in specific hippocampal subregions of VAMP2+/− mice showed significant reductions in potassium-evoked glutamate release. Taken together these studies demonstrate that manipulating the levels of the neurosecretory machinery not only affects neurotransmitter release but also mitigates kindling-induced epileptogenesis.

Hypersensitive glutamate signaling correlates with the development of late-onset behavioral morbidity in diffuse brain-injured circuitry.

In diffuse brain-injured rats, robust sensory sensitivity to manual whisker stimulation develops over 1 month post-injury, comparable to agitation expressed by brain-injured individuals with overstimulation. In the rat, whisker somatosensation relies on thalamocortical glutamatergic relays between the ventral posterior medial (VPM) thalamus and barrel fields of somatosensory cortex (S1BF). Using novel glutamate-selective microelectrode arrays coupled to amperometry, we test the hypothesis that disrupted glutamatergic neurotransmission underlies the whisker sensory sensitivity associated with diffuse brain injury. We report hypersensitive glutamate neurotransmission that parallels and correlates with the development of post-traumatic sensory sensitivity. Hypersensitivity is demonstrated by significant 110% increases in VPM extracellular glutamate levels, and 100% increase in potassium-evoked glutamate release in the VPM and S1BF, with no change in glutamate clearance. Further, evoked glutamate release showed 50% greater sensitivity to a calcium channel antagonist in brain-injured over uninjured VPM. In conjunction with no changes in glutamate transporter gene expression and exogenous glutamate clearance efficiency, these data support a presynaptic origin for enduring post-traumatic circuit alterations. In the anatomically-distinct whisker circuit, the injury-induced functional alterations correlate with the development of late-onset behavioral morbidity. Effective therapies to modulate presynaptic glutamate function in diffuse-injured circuits may translate into improvements in essential brain function and behavioral performance in other brain-injured circuits in rodents and in humans.

Diffuse Brain Injury Elevates Tonic Glutamate Levels and Potassium-Evoked Glutamate Release in Discrete Brain Regions at Two Days Post-Injury: An Enzyme-Based Microelectrode Array Study

Traumatic brain injury (TBI) survivors often suffer from a wide range of post-traumatic deficits, including impairments in behavioral, cognitive, and motor function. Regulation of glutamate signaling is vital for proper neuronal excitation in the central nervous system. Without proper regulation, increases in extracellular glutamate can contribute to the pathophysiology and neurological dysfunction seen in TBI. In the present studies, enzyme-based microelectrode arrays (MEAs) that selectively measure extracellular glutamate at 2 Hz enabled the examination of tonic glutamate levels and potassium chloride (KCl)-evoked glutamate release in the prefrontal cortex, dentate gyrus, and striatum of adult male rats 2 days after mild or moderate midline fluid percussion brain injury. Moderate brain injury significantly increased tonic extracellular glutamate levels by 256% in the dentate gyrus and 178% in the dorsal striatum. In the dorsal striatum, mild brain injury significantly increased tonic glutamate levels by 200%. Tonic glutamate levels were significantly correlated with injury severity in the dentate gyrus and striatum. The amplitudes of KCl-evoked glutamate release were increased significantly only in the striatum after moderate injury, with a 249% increase seen in the dorsal striatum. Thus, with the MEAs, we measured discrete regional changes in both tonic and KCl-evoked glutamate signaling, which were dependent on injury severity. Future studies may reveal the specific mechanisms responsible for glutamate dysregulation in the post-traumatic period, and may provide novel therapeutic means to improve outcomes after TBI.

Potassium-evoked Glutamate Release Liberates Arachidonic Acid from Cortical Neurons

Brain cells in situ contain low concentrations of free polyunsaturated fatty acids such as arachidonic acid (AA) that are released following pathological insults. As a large rise in extracellular [K(+)] accompanies cerebral ischemia, we explored whether this was a stimulus for cellular AA release employing a murine mixed cortical cell culture preparation radiolabeled with AA. Elevating the [K(+)](o) from 5 to 52 mm induced a time-dependent increase in [(3)H]AA release, which reached a plateau after 15 min. Removal of [Ca(2+)](o) or addition of CdCl(2) (100 microm) diminished the net high K(+)-induced AA release, as did treatment of the cultures with tetanus toxin (300 ng/ml) to block endogenous neurotransmitter release. Pharmacological antagonism of both ionotropic and metabotropic glutamate receptors completely prevented high K(+)-evoked AA release, indicating that glutamate was the neurotransmitter in question. Addition of exogenous glutamate mimicked precisely the characteristics of AA release that followed increases in [K(+)](o). Finally, glutamate and AA were released solely from neurons as tetanus toxin did not cleave astrocytic synaptobrevin-2, nor was AA released from pure astrocyte cultures using the same stimuli that were effective in mixed cultures. Taken in toto, our data are consistent with the following scenario: high [K(+)](o) depolarizes neurons, causing an influx of Ca(2+) via voltage-gated Ca(2+) channels. This Ca(2+) influx stimulates the release of glutamate into the synaptic cleft, where it activates postsynaptic glutamate receptors. Events likely converge on the activation of a phospholipase A(2) family member and possibly the enzymes diacylglycerol and monoacylglycerol lipases to yield free AA.

Inhibition of evoked glutamate release by the neuroprotective 5-HT 1A receptor agonist BAY x 3702 in vitro and in vivo

Brain ischemia provoked by stroke or traumatic brain injury induces a massive increase in neurotransmitter release, in particular of the excitotoxin glutamate. Glutamate triggers a cascade of events finally leading to widespread neuronal cell damage and death. The aminomethylchroman derivative BAY x 3702 is a novel neuroprotectant which shows pronounced beneficial effects in various animal models of ischemic brain injury. As shown previously BAY x 3702 binds to 5-HT 1A receptors of different species in subnanomolar range and is characterized as a full receptor agonist. In this study we investigated the influence of BAY x 3702 on potassium-evoked glutamate release in vitro and ischemia-induced glutamate release in vivo. In rat hippocampal slices BAY x 3702 inhibited evoked glutamate release in a dose-dependent manner (IC 50=1 μM). This effect was blocked by the selective 5-HT 1A receptor antagonist WAY 100635, indicating that BAY x 3702 specifically acts via 5-HT 1A receptors. In vivo, release of endogenous aspartate and glutamate was measured in the cortex of rats by microdialysis before and after onset of permanent middle cerebral artery occlusion. Single dose administration of BAY x 3702 (1 μg/kg or 10 μg/kg i.v.) immediately after occlusion reduced the increase and total release of extracellular glutamate by about 50% compared to non-treated animals, whereas the extracellular aspartate levels were not significantly affected. Inhibition of glutamate release may therefore contribute to the pronounced neuroprotective efficacy of BAY x 3702 in various animal models of ischemic brain damage.

Fetal Ventral Mesencephalic Grafts Functionally Reduce the Dopamine D2 Receptor Supersensitivity in Partially Dopamine Reinnervated Host Striatum

Grafting of ventral mesencephalic tissue in Parkinson's disease results in a partial dopaminergic reinnervation of host brain and dopamine agonist-induced rotational behavior is not completely reversed. To study a possible malfunction of the grafts, extracellular recordings with local applications of quinpirole were utilized and the neurophysiological results showed that a normalization of the upregulated dopamine D2 receptor supersensitivity occurred in reinnervated areas of the host striatum as well as in noninnervated areas remote from the graft innervation. Furthermore, the inhibitory effects on striatal nerve cell firing rate by the D1 receptor agonist SKF 81297 were not different in noninnervated or reinnervated areas of the striatum compared to the control side as seen from the dose–response curves. However, spontaneous striatal neuronal firing was significantly upregulated in noninnervated areas, while it was normalized in areas reached by graft-derived nerve fibers. Dual-probe microdialysis studying potassium-evoked glutamate release revealed that there was no difference in extracellular glutamate levels measured within or lateral to graft dopamine reinnervation. Thus, the upregulated spontaneous activity was not due to a difference in extracellular glutamate levels. The remaining rotational behavior seen after grafting was studied and recordings were performed in the striatum following systemic injection of the D1/D2 agonist apomorphine. The results revealed that apomorphine at the dose used to elicit turning behavior (0.05 mg/kg) still affected striatal neurons in noninnervated areas, while no effect was detected in reinnervated areas and in the intact side. However, a lower dose of apomorphine (0.005 mg/kg) showed no effects on striatal firing in graft reinnervated striata but only after dopamine depletion. In conclusion, the D2 supersensitivity is downregulated in graft-reinnervated striatum as well as in striatal areas lateral to the reinnervation when using selective D2 agonists, but the downregulation is not completely normalized when studying combined effects of D1/D2 agonists. Furthermore, the striatal neurons were firing significantly faster in noninnervated areas compared to reinnervated areas of graft-reinnervated striatum, which was most likely not due to changes in the glutamatergic input.

Neuropeptide Y inhibits potassium-stimulated glutamate release through Y2 receptors in rat hippocampal slices in vitro.

1. We investigated the effects of neuropeptide Y (NPY), peptide YY (PYY), NPY13-36, NPY18-36, [Leu31][Pro34]NPY and of pancreatic polypeptide Y (PPY) on calcium-dependent, potassium-stimulated glutamate release in superfused rat hippocampal slices. 2. NPY, PYY and the Y2 receptor agonist NPY13-36 equipotently inhibited the release of glutamate. The half-maximal response was observed at about 10 nM in a dose-dependent manner (3 to 100 nM). Maximal inhibition of 50 to 60% was obtained at 100 nM. At higher concentrations of the peptides (300 nM and 1 microM) this inhibition was partially or entirely reversed. Porcine NPY13-36 and NPY18-36 inhibited glutamate release by about 44% at 100 nM. 3. The specific Y1 receptor agonist, [Leu31][Pro34]NPY, caused an insignificant increase in glutamate release at 100 to 300 nM concentrations. PPY had no effect on potassium-evoked glutamate release in hippocampal slices at concentrations of 30 nM to 1 microM. 4. The experiments support previous electrophysiological data. They suggest a potent inhibitory action of NPY through NPY-Y2 receptors on the release of the excitatory amino acid glutamate in rat hippocampus. Especially under conditions of increased NPY synthesis, such as in epilepsy, this mechanism may be of pathophysiological relevance.

Extracellular brain glutamate during acute liver failure and during acute hyperammonemia simulating acute liver failure: An experimental study based on in vivo brain dialysis

Hyperammonemia is thought to be important in the pathogenesis of hepatic encephalopathy. However, the mechanism leading to ammonia toxicity is still not known. Since the metabolism of the most important excitatory neurotransmitter, glutamate, is closely linked to that of ammonia, it has been postulated that hyperammonemia lowers the availability of the neurotransmitter glutamate. To study this hypothesis, we used brain dialysis to measure glutamate levels in extracellular cerebral fluid from rabbits with acute ischemic liver failure or acute hyperammonemia. The basal glutamate concentration was found to be increased during both acute liver failure (start of experiments 4.9 +/- 1.7 mumol/l; end of experiments 9.5 +/- 2.1 mumol/l, n = 6, difference p < 0.05) and acute hyperammonemia (start of experiments 4.4 +/- 1.2 mumol/l; end of experiments 7.3 +/- 1.8 mumol/l, n = 7, difference p > 0.05) (mean +/- SEM). Both the veratridine- and the potassium-evoked glutamate release were increased during acute liver failure but appeared normal during hyperammonemia. We conclude that during acute liver failure and acute hyperammonemia in the rabbit there is no decreased glutamate availability in the extracellular space of the cortical brain; on the contrary, we found evidence for increased extracellular glutamate concentrations in the cortical brain, which were more pronounced in acute liver failure. Experimental hepatic encephalopathy is thus not due to cerebral glutamate deficiency.

Extracellular glutamate levels increase with age in the lateral striatum: Potential involvement of presynaptic D‐2 receptors

In the lateral striatum of aged rats, dopamine D-2 receptor density is reduced and glutamate tissue content is elevated. D-2 receptor agonists have been shown to inhibit stimulated glutamate release. In the present study, microdialysis was used to investigate a potential role for D-2 receptors in the modulation of striatal glutamate efflux from 4-, 12-, 18-, and 24-26-month-old Fischer 344 rats. Extracellular basal glutamate concentrations significantly increased as a function of age in the lateral, but not medial, striatum. Neither the D-2 agonist, LY 163502, nor the D-2 antagonist, sulpiride, influenced basal glutamate efflux, suggesting that the dopaminergic system is not involved in the observed age-related increase in extracellular basal glutamate levels. In contrast to basal efflux, potassium-evoked glutamate release was not altered with age. However, LY 163502 significantly inhibited stimulated glutamate release in 4-month-old rats. This inhibitory action was not observed at any other age. Sulpiride alone did not alter stimulated glutamate release, but it did block the inhibitory effect of LY 163502 in the 4-month-old rats. These results provide in vivo evidence for an age-related functional loss in the modulation of striatal glutamate release by dopamine D-2 receptors in addition to increased basal glutamate efflux, which is not related to D-2 receptor modulation. Such mechanisms could be important in the pathophysiology of striatal cell death during aging and age-related neurodegenerative diseases.

Glutamate in the inferior colliculus plays a critical role in audiogenic seizure initiation

Alterations of excitant amino acid (EAA) action are implicated in seizure susceptibility in the genetically epilepsy-prone rat (GEPR). The inferior colliculus (IC) is critical for audiogenic seizure (AGS) initiation in the GEPR. The present study observed that bilateral microinjection into the IC of l-canaline, a glutamate synthesis inhibitor, decreased AGS severity in the GEPR and also decreased potassium-evoked release of glutamate from IC slices. Bilateral microinjection of NMDA receptor antagonists, 2-amino-7-phosphonoheptanoate (AP7) or 3-((±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) into IC blocked AGS, and an antagonist at non-NMDA EAA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), also blocked AGS. NMDA receptor antagonists were 5–200 times more effective than CNQX. Microinjection of a non-competitive NMDA receptor antagonist, dizocilpine (MK-801), into IC had little effect except with very high doses. Microinjection of CPP or AP7 into the IC blocked AGS at considerably lower doses as compared to pontine reticular formation (PRF). However, MK-801 attenuated AGS when microinjected into PRF at doses that were ineffective in IC. Systemically administered CPP blocked AGS and significantly reduced IC neuronal firing in the behaving GEPR, suggesting an important action of systemically administered NMDA receptor antagonists on brainstem auditory nuclei critical to AGS. The present results support a critical role for glutamate acting, in part, through NMDA receptors in IC in initiation of AGS.

Glutamate, of the endogenous primary alpha-amino acids, is specifically released from hair cells by elevated extracellular potassium.

A hair cell (octavolateralis mechanoreceptor cell) sheet preparation from the trout saccular macula was superfused with bicarbonate-based physiological saline. Among the primary amine-containing compounds resolved by cation-exchange HPLC, glutamate alone was released in a statistically significant manner with elevation of extracellular [K+] from 3.5 to 14 mM in the presence of 1.8 mM calcium. Release of glutamate averaged 10.9 +/- 2.5 pmol (mean +/- SEM) over a 10-min period for a hair cell sheet preparation representing 20 micrograms of cell protein. No potassium-evoked release of glutamate was observed in 0 mM calcium/10 mM magnesium saline, suggesting calcium dependency. Because the sheet preparation, by the method of its isolation, contained only the hair cell as the intact cell type, release of glutamate, induced by relatively small increases in extracellular potassium, can be attributed directly to the receptor cell. The specific release of glutamate and its block by magnesium are consistent with the hypothesis that glutamate is one neurotransmitter/neuromodulator mediating receptoneural transmission in the octavolateralis periphery.

Clonidine inhibition of potassium-evoked release of glutamate and aspartate from rat cortical synaptosomes

Release of endogenous glutamic acid (Glu), aspartic acid (Asp) and γ-aminobutyric acid (GABA) has been investigated using synaptosomes prepared from rat cerebral cortex. Exposure in superfusion to a depolarizing concentration of KCl (30 mM) evoked 3-, 2- and 2-fold increases in Glu, Asp adn GABA release, respectively. More than 70% of Glu and Asp overflow were calcium-dependent, although 67% of the GABA overflow was calcium-independent. Clonidine inhibited the K +-evoked overflow of Glu and Asp in a concentration-dependent manner, but the GABA overflow was not inhibited. Clonidine inhibited K +-evoked Glu and Asp overflow to 40 and 30% of the control with a potency (IC 50) of 11 and 36 nM, respectively. Similarly, norepinephrine inhibited the K +-evoked overflow of Glu and Asp, although phenylephrine and isoproterenol showed no effect. Rauwolscine, yohimbine and idazoxan counteracted the effects of clonidine on Glu and Asp overflow. The data suggest that the depolarization-evoked overflow of excitatory amino acids is regulated in an inhibitory fashion byα 2 adrenoceptors, which are located on the nerve terminals of Glu and Asp neurons in rat cortex.

Enhanced aspartate release from hippocampal slices of epileptic (El) mice.

The release of putative neurotransmitters [aspartate, glutamate, and gamma-aminobutyric acid (GABA)] was studied in hippocampal slices from adult normal C57BL/6J (B6) and El (epileptic) mice. The El mice, a genetic model of temporal lobe epilepsy, had an average of 86 seizures. Sets of B6 and El hippocampal slices (400 microns thick) were incubated in a series of normal and high potassium (60 mM) buffers in the presence or absence of calcium. The calcium-dependent and calcium-independent potassium-induced release of amino acids was compared in each mouse strain. Release of endogenous amino acids was measured using liquid chromatography with electrochemical detection and was expressed as picomoles of amino acid released per milliliter of incubation buffer per minute of incubation per slice +/- SEM. No significant differences were found between the El and B6 mice for the calcium-dependent potassium-evoked release of glutamate (18.20 +/- 2.62 and 15.41 +/- 3.56), or GABA (17.28 +/- 2.90 and 12.73 +/- 1.37), respectively. Aspartate release, however, was significantly higher in the El mice (6.62 +/- 0.69) than in the B6 mice (3.31 +/- 0.72). These findings suggest that enhanced aspartate release may be related to seizure expression in El mice.